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• W2146705615 abstract "Ribose-5-phosphate isomerase (Rpi) acts as a key enzyme in the oxidative and reductive pentose-phosphate pathways for the conversion of ribose-5-phosphate (R5P) to ribulose-5-phosphate and vice versa. We have determined the crystal structures of Rpi from Thermus thermophilus HB8 in complex with the open chain form of the substrate R5P and the open chain form of the C2 epimeric inhibitor arabinose-5-phosphate as well as the apo form at high resolution. The crystal structures of both complexes revealed that these ring-opened epimers are bound in the active site in a mirror symmetry binding mode. The O1 atoms are stabilized by an oxyanion hole composed of the backbone amide nitrogens in the conserved motif. In the structure of the Rpi·R5P complex, the conversion moiety O1-C1-C2-O2 in cis-configuration interacts with the carboxyl oxygens of Glu-108 in a water-excluded environment. Furthermore, the C2 hydroxyl group is presumed to be highly polarized by short hydrogen bonding with the side chain of Lys-99. R5P bound as the ring-opened reaction intermediate clarified the high stereoselectivity of the catalysis and is consistent with an aldose-ketose conversion by Rpi that proceeds via a cis-enediolate intermediate. Ribose-5-phosphate isomerase (Rpi) acts as a key enzyme in the oxidative and reductive pentose-phosphate pathways for the conversion of ribose-5-phosphate (R5P) to ribulose-5-phosphate and vice versa. We have determined the crystal structures of Rpi from Thermus thermophilus HB8 in complex with the open chain form of the substrate R5P and the open chain form of the C2 epimeric inhibitor arabinose-5-phosphate as well as the apo form at high resolution. The crystal structures of both complexes revealed that these ring-opened epimers are bound in the active site in a mirror symmetry binding mode. The O1 atoms are stabilized by an oxyanion hole composed of the backbone amide nitrogens in the conserved motif. In the structure of the Rpi·R5P complex, the conversion moiety O1-C1-C2-O2 in cis-configuration interacts with the carboxyl oxygens of Glu-108 in a water-excluded environment. Furthermore, the C2 hydroxyl group is presumed to be highly polarized by short hydrogen bonding with the side chain of Lys-99. R5P bound as the ring-opened reaction intermediate clarified the high stereoselectivity of the catalysis and is consistent with an aldose-ketose conversion by Rpi that proceeds via a cis-enediolate intermediate. Ribose-5-phosphate isomerase (Rpi 1The abbreviations used are: Rpiribose-5-phosphate isomerasettRpiThermus thermophilus rpiR5Pribose-5-phosphateA5Parabinose-5-phosphateRu5Pribulose-5-phosphateTIMtriosephosphate isomerasePGIphosphoglucose isomeraseSeMetselenomethionineIPP2-(N-formyl-N-hydroxy)-aminoethylphosphateDHAPdihydroxyacetone phosphate.; EC 5.3.1.6) is ubiquitous throughout all living cells and highly conserved in amino acid sequences. Rpi acts as a key enzyme in the oxidative pentose-phosphate cycle where it catalyzes the reversible conversion of ribose-5-phosphate (R5P) to ribulose-5-phosphate (Ru5P). Rpi additionally plays a central role in the reductive pentose-phosphate cycle (Calvin cycle) of photosynthetic organisms. Rpi catalyzes the final step of the conversion of glucose-6-phosphate into ribose-5-phosphate, which is required for the synthesis of nucleotides. It also converts R5P to Ru5P in the final step to regenerate ribulose-1,5-bisphosphate as the acceptor of CO2 in the Calvin cycle. In the non-oxidative pathway of the pentose-phosphate cycle, R5P is the precursor of 5-phosphoribosylpyrophosphate, which is utilized for syntheses of amino acids such as histidine and tryptophan, purine and pyrimidine nucleotides, and NAD (1.Hove-Jensen B. J. Bacteriol. 1988; 170: 1148-1152Crossref PubMed Google Scholar), whereas Ru5P is the riboflavin precursor (2.Volk R. Bacher A. J. Biol. Chem. 1991; 266: 20610-20618Abstract Full Text PDF PubMed Google Scholar). In the Calvin cycle, it has been shown that Rpi forms a functional multienzyme complex with five other enzymes, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), to catalyze the consecutive reactions on chloroplast thylakoid membranes in situ (3.Suss K.H. Arkona C. Manteuffel R. Adler K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5514-5518Crossref PubMed Scopus (131) Google Scholar). The direction of the reaction catalyzed by Rpi is essentially driven by the R5P and Ru5P concentrations. ribose-5-phosphate isomerase Thermus thermophilus rpi ribose-5-phosphate arabinose-5-phosphate ribulose-5-phosphate triosephosphate isomerase phosphoglucose isomerase selenomethionine 2-(N-formyl-N-hydroxy)-aminoethylphosphate dihydroxyacetone phosphate. The catalytic mechanism of Rpi (Fig. 1A) is believed to initiate with a ring opening of the substrate followed by isomerization of the open chain form. Isotope exchange studies have suggested that the isomerization between ketose and aldose by triosephosphate isomerase (TIM), phosphoglucose isomerase (PGI), and Rpi involves a proton transfer between the C1 and C2 positions of the substrate via the cis-enediol(ate) intermediate (4.Rose I.A. Edward L.O.C. Biochim. Biophys. Acta. 1960; 42: 159-160Crossref PubMed Scopus (51) Google Scholar, 5.Topper Y.J. J. Biol. Chem. 1956; 225: 419-425Abstract Full Text PDF Google Scholar). The transferred proton in catalysis remains on the same side of the plane in the enediol(ate) structure (6.Fersht A. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman, New York1999: 251-252Google Scholar). Then, the proton abstracted by a single base is given back to the C1 or C2 position, resulting in the conversion of Ru5P to R5P and vice versa (Fig. 1A). Recently, the crystal structures of Escherichia coli Rpi in both the apo form and in complex with the β-anomer furanose form of arabinose-5-phosphate (A5P) have been determined (7.Rangarajan E.S. Sivaraman J. Matte A. Cygler M. Proteins. 2002; 48: 737-740Crossref PubMed Scopus (30) Google Scholar, 8.Zhang R. Andersson C.E. Savchenko A. Skarina T. Evdokimova E. Beasley S. Arrowsmith C.H. Edwards A.M. Joachimiak A. Mowbray S.L. Structure. 2003; 11: 31-42Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Based on this structure, a ring-opening mechanism was proposed. In addition, the crystal structures of a tetrameric Rpi from the hyperthermophilic bacterium Pyrococcus horikoshii in the apo form and in complex with the inhibitor 4-phosphoerythronic acid have also been reported and have elucidated aspects of the thermostability of this protein (25.Ishikawa K. Matsui I. Payan F. Cambillau C. Ishida H. Kawarabayasi Y. Kikuchi H. Roussel A. Structure. 2002; 10: 877-886Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). However, to date, there is little structural information on the aldose-ketose isomerization mechanism of Rpi. Here we report on the first crystal structures of Rpi from the extreme thermophile, Thermus thermophilus HB8, in complex with the ring-opened form of substrate R5P at 2-Å resolution and the open chain form of the C2 epimer inhibitor A5P (Fig. 1B) at 1.7-Å resolution (hereafter referred to as the ttRpi·R5P and ttRpi·A5P complexes, respectively) as well as the apo form at 1.8-Å resolution. The high resolution crystal structure of ttRpi·R5P complex suggests that the isomerization of Rpi proceeds via a cis-enediolate intermediate with the negatively charged O1 (Fig. 1C) stabilized by the oxyanion hole. Protein Expression and Purification—Rpi with selenium-substituted methionine (SeMet) was prepared for structural determination by multiwavelength anomalous dispersion (MAD) phasing. The plasmid pET-11a (Novagen), which carries a gene encoding ttRpi under the T7 promoter, was supplied from the RIKEN Structurome Project (Kuramitsu and Yokoyama). The protein was over-expressed in the methionine auxotrophic E. coli strain B834(DE3)plysS (Novagen) grown in LeMaster medium with SeMet. After cell disruption (14 g) and removal of debris by centrifugation, the supernatant was heat-treated at 70 °C in 11.5 min. Then, ttRpi was purified with the SuperQ Toyopearl (Tosoh), Resource Q (Amersham Biosciences), and hydroxyapatite (Bio-Rad) column chromatography and followed by a Superdex 75 column (Amersham Biosciences). Twenty-nine milligrams of purified protein was obtained. The sample showed a single band at 24 kDa on a 12.5% SDS-PAGE, and was also verified with N-terminal sequence analysis. Rpi in solution was detected as a homo-dimer with Superose 12 (Amersham Biosciences) gel filtration and dynamic light scattering (Protein Solution) analysis. The native proteins used in the R5P-bound complex were purified and verified in a similar way as the SeMet proteins. Enzymatic Characterization—Reversible isomerization (R5P to Ru5P) enzymatic activity was observed from the absorption changes at 290 nm as described previously by Wood (9.Wood T. Anal. Biochem. 1970; 33: 297-306Crossref PubMed Scopus (34) Google Scholar). The reaction mixture contains 5 nm Rpi, 0.1 m NaCl, and 50 mm Hepes (pH 7.5). The increase of absorption was measured using R5P (Fluka) ranging from 0.5 to 30 mm at 50 °C. The kinetic parameters were calculated from Lineweaver-Burk plots and obtained by averaging three independent measurements. The inhibition by A5P was assayed at 50 °C in assay solution containing the various concentrations of A5P. The Ki value was calculated from Dixon plots. Crystallization and Data Collections—The screening of the crystallization conditions of SeMet Rpi was performed with the oil batch method (10.Chayen N.E. Shaw-Stewart P.D. Maeder D.L. Blow D.M. J. Appl. Crystallogr. 1991; 23: 297-302Crossref Google Scholar) using the recently developed fully automatic protein crystallization and observation system “TERA” installed at the Highthroughput Factory at RIKEN Harima institute (11.Sugahara M. Miyano M. Tanpakushitsu Kakusan Koso. 2002; 47: 1026-1032PubMed Google Scholar). The crystals were obtained from a solution containing 10 mg/ml protein, 27.5% polyethylene glycol 4000, 0.1 m Tris-HCl (pH 8.4), and 1 m LiCl. The crystals grew within 1 week at 18 °C. Crystals were flash-cooled in the cryoprotectant 20% (v/v) glycerol. X-ray diffraction data of the crystals was collected at 100 K up to 1.8 Å resolution using a charged coupled area detector (Rigaku Jupitor 210) at the beam line BL45XU-PX (12.Yamamoto M. Kumasaka T. Fujisawa T. Ueki T. J. Synchrotron Radiat. 1998; 5: 222-225Crossref PubMed Scopus (51) Google Scholar) at SPring-8. The wavelength was set to 0.900 Å (remote), 0.9791 Å (peak), and 0.9795 Å (edge) with a crystal-detector distance of 200 mm. The crystals belonged to the space group C2221 with the unit-cell parameters a = 62.10 Å, b = 61.97 Å, and c = 131.34 Å and one molecule in the asymmetric unit with a Vm value (13.Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7926) Google Scholar) of 2.6 Å3/Da. Crystals of R5P- or A5P-bound Rpi were obtained after soaking native or SeMet derivative crystals in solution containing 10 mm R5P (Fluka) or 10 mm A5P (Sigma), for 14 h at 15 °C. Data collections of two complexes were performed at a wavelength of 1 Å. The crystals of ttRpi·R5P and ttRpi·A5P complexes were isomorphous compared with unsoaked crystal and diffracted to 2- and 1.7-Å resolution, respectively. All diffraction images were processed, integrated, and scaled using the program HKL2000 (14.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38609) Google Scholar). Structural Determination and Refinement—All selenium sites except for the N-terminal were found and refined, and initial phases were calculated with the programs SOLVE (15.Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1872-1877Crossref PubMed Scopus (64) Google Scholar) and RESOLVE (16.Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1755-1762Crossref PubMed Scopus (166) Google Scholar). Initial model building was carried out automatically by ARP/wARP (17.Morris R.J. Perrakis A. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 968-975Crossref PubMed Scopus (221) Google Scholar), and additional model building was manually performed with O (18.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Refinement of the model was carried out using CNS (19.Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. GrosseKunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). Water molecules were assigned during refinement, and chloride ions were identified as judged by peak heights and positions in the electron density map. The final model of the apo form has a working R factor of 19.2% and a free R factor of 21.5% at 1.8-Å resolution. Ten percent of the data set was selected for free R factor calculations. The crystal structures of the ttRpi·R5P and ttRpi·A5P complexes were solved by the molecular replacement method with the program AMoRe (20.Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) using the apo form structure as the search model. The ligand models generated using the program Quanta (Accelrys) were fitted initially to electron density maps with coefficients Fo–Fc. Refinement of the ttRpi·R5P complex was performed at 2-Å resolution with a working R factor of 18.8% and a free R factor of 23.9%. The ttRpi·A5P complex was finally refined at a resolution of 1.7 Å to a working R factor of 20.0% and a free R factor of 21.5%. The model quality for the three structures was verified by the program PROCHECK (21.Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), which showed that all the main chain torsion angles were within the allowed regions. A summary of the statistics for structural determination is given in Table I. Fig. 2, A and B as well as Fig. 4, A and C and Figs. 5 and 6 were made using the programs MOLSCRIPT (22.Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and Raster3D (23.Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2859) Google Scholar).Table IData collection, structure determination, and refinement statisticsApo form (remote)Apo form (peak)Apo form (edge)R5P complexA5P complexData Wavelength (Å)0.9000 (remote)0.9791 (peak)0.9795 (edge)11.01 Resolution (Å)1.801.801.8021.74 Space groupC2221C2221C2221 Unit-cell parametersa (Å)62.1061.8962.35b (Å)61.9762.0162.54c (Å)131.34131.17131.25 No. of reflectionsObserved224,876189,398190,790120,105194,173Unique23,90923,01023,02316,93126,688 Completeness (%)aNumbers in parentheses refer to data for the high resolution outer shell. The resolution ranges of their outer shells are 1.88-1.80 Å for the apo form, 2.09-2 Å for the R5P complex, and 1.82-1.74 Å for the A5P complex.99.8 (98.1)96.1 (76.5)96.1 (76.4)97.7 (82.6)100 (100) I/σaNumbers in parentheses refer to data for the high resolution outer shell. The resolution ranges of their outer shells are 1.88-1.80 Å for the apo form, 2.09-2 Å for the R5P complex, and 1.82-1.74 Å for the A5P complex.29.5 (7.3)23.2 (5.2)19.3 (3.4)42.7 (30.1)24.1 (7.7) Rmerge (%)aNumbers in parentheses refer to data for the high resolution outer shell. The resolution ranges of their outer shells are 1.88-1.80 Å for the apo form, 2.09-2 Å for the R5P complex, and 1.82-1.74 Å for the A5P complex.,bRmerge = ΣI - 〈I〉/Σ I.7.0 (22.5)7.8 (27.4)9.7 (42.2)4.2 (6.7)7.7 (34.1) FOM (SOLVE/RESOLVE)cFOM, figure of merit.0.59/0.65Structural refinement Resolution (Å)43.9-1.8050.0-2.0044.2-1.74 No. of residues225 (3-227)225 (3-227)225 (3-227) No. of ions (chloride)322 No. of waters249286310 Rcryst (%)aNumbers in parentheses refer to data for the high resolution outer shell. The resolution ranges of their outer shells are 1.88-1.80 Å for the apo form, 2.09-2 Å for the R5P complex, and 1.82-1.74 Å for the A5P complex.19.2 (21.3)18.8 (20.4)20 (26.5) Rfree (%)aNumbers in parentheses refer to data for the high resolution outer shell. The resolution ranges of their outer shells are 1.88-1.80 Å for the apo form, 2.09-2 Å for the R5P complex, and 1.82-1.74 Å for the A5P complex.,dRcryst and Rfree = Σ||Fo - Fc||/ΣFo, where the free reflections (10% of the total used) were held aside for Rfree throughout refinement.21.5 (23.4)23.9 (25.9)21.5 (27.5) R.m.s. deviationBonds (Å)0.0050.0050.005Angles (°)1.31.31.4 Average B factors (Å2)Protein11.112.914.3Ligand1627.8Ion1414.417.8Waters26.829.131.1 Ramachandran plotMost favored (%)94.494.494.9Allowed (%)5.65.65.1a Numbers in parentheses refer to data for the high resolution outer shell. The resolution ranges of their outer shells are 1.88-1.80 Å for the apo form, 2.09-2 Å for the R5P complex, and 1.82-1.74 Å for the A5P complex.b Rmerge = ΣI - 〈I〉/Σ I.c FOM, figure of merit.d Rcryst and Rfree = Σ||Fo - Fc||/ΣFo, where the free reflections (10% of the total used) were held aside for Rfree throughout refinement. Open table in a new tab Fig. 4Active site architecture of Rpi.A, close-up view of the active site in the apo form of ttRpi. Representations of three ligand-binding motifs with the side chains of the active site residues are shown as stick models colored purple. Water molecules are shown as red spheres labeled with a numbered W. The chloride ion (magenta) that binds to the oxyanion hole (yellow) is shown with the σA weighted Fo - Fc electron density map (5 σ) calculated after the omission of the relevant moiety from the model at a resolution of 1.8 Å. The hydrogen-bonding interactions within 3.4 Å are represented by black broken lines, whereas the interactions involving the chloride ion and salt bridges between Glu-108 and Lys-99 are represented by orange and green broken lines, respectively. B, close-up view of the active site of the ttRpi·R5P complex. The model of R5P is shown with the Fo - Fc omit electron density map with a 3 σ counter level in light blue and a 6 σ counter level in green at a resolution of 2 Å. The hydrogen-bonding interactions between R5P and the active site residues and between the active site residues themselves are shown as the thick and thin broken lines in black, respectively. Interactions involving O1 of R5P are indicated as orange broken lines. C, close-up view of the active site of ttRpi·A5P complex. The model of A5P is shown with the omit electron density map and coefficients of Fo - Fc with a 2.8 σ counter level in light blue and a 6 σ counter level in green at a resolution of 1.7 Å. D, schematic representations of interactions in the active site of the apo form of ttRpi with bond lengths. The distance of hydrogen bonds is discriminated by color representation (cyan, within 2.8 Å; blue, between 2.9 and 3.3 Å; and magenta, longer than 3.4 Å). The interactions with the chloride ion are shown in orange broken lines. The salt bridges between Lys-99 and Glu-108 are shown with green broken lines. E, schematic view of active site interactions of ttRpi·R5P complex. F, schematic view of interactions in the ttRpi·A5P complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Representation of the overlay between the bound R5P and A5P, showing the mirror binding mode of the two ligands. The various broken lines show the interactions between the residues and R5P as the cis-enediolate intermediate. The orange broken lines show the interactions between O1 and the oxyanion hole as described in the text. The black broken lines include the hydrogen bonding interactions involved in Asp-86, Asp-89, and Lys-99. The interactions by Glu-108 are shown as thick red broken lines.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Comparison of the active sites of ttRpi·R5P complex (cyan) and the two TIM complexes, TIM·DHAP (magenta) and TIM·IPP (green). In the Rpi active site the interactions of two important catalytic residues, Glu-108 and Lys-99, with R5P (red broken line) and a representation of the oxyanion hole (yellow) are shown. Interactions of DHAP and several active site residues (Asn-10, Lys-12, His-95, and Glu-165) of TIM (magenta) are shown as black broken lines. The corresponding residues of the TIM·IPP complex (light green) are overlaid with the TIM·DHAP complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Characterization of ttRpi—The ttRpi sample used for crystallization was shown to exhibit normal enzyme activity. The kinetic parameters of ttRpi in the forward reaction using R5P as the substrate at 50 °C were kcat of 1072 ± 78 s-1, Km of 1.63 ± 0.24 m, and kcat/Km of (6.64 ± 0.91) × 105m-1 s-1. The Ki value was 0.89 ± 0.14 mm. The enzyme characteristics are consistent with those reported previously for E. coli and spinach Rpis. Overall Structure of ttRpi—The crystal structure demonstrated that ttRpi exists as a homo-dimer (Fig. 2A), which is consistent with the molecular weights determined for the protein in solution by both gel filtration (Mr = 47,300) and dynamic light scattering measurement (Mr = 50,000). The two monomers are related by a crystallographic 2-fold symmetry. The close interaction between the two monomers is stabilized by two chloride ions (Fig. 2A). There are also many hydrophobic interactions, four side chain salt bridges between the side chains of Arg-193 and Glu-197 and between Asp-75 and Arg-146, and a water molecule-mediated hydrogen bond network. The buried solvent-accessible surface is 2,547 Å2 as calculated using the program GRASP (24.Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5318) Google Scholar). Each monomer of ttRpi (Fig. 2B) is composed of two α/β domains with dimensions of ∼45 Å × 35 Å × 25 Å as seen in E. coli and P. horikoshii Rpis (7.Rangarajan E.S. Sivaraman J. Matte A. Cygler M. Proteins. 2002; 48: 737-740Crossref PubMed Scopus (30) Google Scholar, 8.Zhang R. Andersson C.E. Savchenko A. Skarina T. Evdokimova E. Beasley S. Arrowsmith C.H. Edwards A.M. Joachimiak A. Mowbray S.L. Structure. 2003; 11: 31-42Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 25.Ishikawa K. Matsui I. Payan F. Cambillau C. Ishida H. Kawarabayasi Y. Kikuchi H. Roussel A. Structure. 2002; 10: 877-886Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The larger domain (residues 3–130 and 206–227) comprises a seven-stranded mixed β-sheet formed by six parallel (S3, S2, S1, S4, S7, and S13) and one anti-parallel β-strands (S14), four α helices (H1–4), and a three-stranded anti-parallel β-sheet (S5, S6, and S12). The active site is located within the larger domain with the architecture formed by loops S1-H2, S4-S5, and S6-H4 and H4. The small domain consists of a four-stranded, anti-parallel β-sheet (S9, S11, S8, and S12) and two helices (H5 and H6). The structure of the larger domain of ttRpi is overlaid with those of E. coli and P. horikoshii with root mean square deviations of 1.3 Å for 140 Cα and 1.2 Å for 150 Cα, respectively. When the larger domains are superimposed, the small domain of ttRpi moves substantially with respect to the larger domain by a 3-Å shift of the H5 helix. The Active Site Architecture with an Oxyanion Hole—Three highly conserved motifs (Fig. 2C) form the active site, i.e. the phosphate (P) and sugar (S) recognition motifs as well as the catalytic (C) motif over all the organisms. The P-, S-, and C-motifs are located in the loops of S1-H2, S4-S5, and S6-H4, respectively (Fig. 2, B and C). The active cavity is shallow and binds the dehydrated pentose-phosphates without any water molecules around C1 to C3 in both complex structures (Figs. 3 and 4, B and C). Most of the molecular surfaces of the ligand binding cavity are negatively charged with a noticeable positive patch formed by Lys-9 and Lys-126, which are important for allowing accommodation of the negatively charged phosphate moiety (Fig. 3). In the apo ttRpi structure, a chloride ion (Cl- in Fig. 2B) is bound at the bottom of the active site cavity. The side chains of the residues positioned within the active site, including Asp-86, Asp-89, Lys-99, Glu-108, and Glu-112, form hydrogen bond networks with 10 bound water molecules (Fig. 4, A and D). The bound chloride anion interacts with five backbone amide nitrogens (Gly-100 to Leu-105) located at the center of the C-motif, the most conserved stretch of amino acids among the Rpis. Distances between the chloride anion and the amide nitrogens are in the range of 3.3–4.2 Å. Thus, this loop makes an oxyanion hole to stabilize the negatively charged atoms of the reaction intermediate during catalysis (Fig. 1A). Moreover, both the carboxyl oxygen atoms of Glu-108 form a bidentate interaction with the chloride ion (3 Å and 3.7 Å). Bound R5P Interaction as Reaction Intermediate—In the structure of ttRpi·R5P complex, an extended ring-opened form of R5P provided the best fit to the electron density. This conformation differs from the ring-closed furanose form of A5P observed in the E. coli Rpi·A5P complex structure (Figs. 3 and 4, B and E). The torsion angle of O1-C1-C2-O2 of R5P is almost 0°, and it may represent the cis-enediolate intermediate (Fig. 1A). Contrarily, that of A5P is 93°. However, the possibility cannot be excluded that other forms, including aldose and/or ketose in addition to the enediolate, contribute to the electron density map of R5P, because we could not determine very accurate geometric parameters of the form of R5P from the 2-Å resolution data due to the model bias of the crystallographic refinement. In the best refined current model, the C1-O1, C2-O2, and C1-C2 bond lengths of R5P are 1.37, 1.39, and 1.51 Å, respectively, whereas those of A5P are 1.19, 1.38, and 1.61 Å. The atoms of O1-C1-C2(-O2)-C3 of R5P are in a plane with the bond angles of C1-C2-C3 (113°), C1-C2-O2 (124°), and C3-C2-O2 (123°), suggesting that the C2 orbital acquires hybridization between sp2 (120°) and sp3 (109.5°), whereas those of A5P are obviously in off-plane with the bond angles of C1-C2-C3 (115°), C1-C2-O2 (108°), and C3-C2-O2 (108°). The O1 atom of the bound R5P displaces the chloride ion from the oxyanion hole with distances between backbone amide nitrogens and O1 of 3.5 Å (Gly-100), 3.5 Å (Gly-102), 3.4 Å (Gly-103), 3 Å (Ala-104), and 3.6 Å (Leu-105). A prominent feature of the interaction between ttRpi and R5P is that the O1 of R5P should carry either a full or partial negative charge. Another notable feature is the interaction between both the carboxyl groups of Glu-108 and O1-C1-C2-O2 of the bound R5P. The Oϵ2 carboxyl oxygen of Glu-108 is unusually close to both C1 (3.3 Å) and C2 (3.2 Å), whereas Oϵ1 forms hydrogen bond interactions with O1 (3.1 Å) and O2 (2.8 Å) (Fig. 4E). The O2 oxygen of the bound R5P forms the short hydrogen bond with the side chain of Lys-99 (2.5 Å) as well as the backbone amide nitrogen of Gly-100 (3.0 Å). The distances between atoms indicate a tight C–H···O hydrogen bond between C1-C2 and the syn orbital of Oϵ2, including the putative hydrogen attached to C2 of R5P. This close interaction suggests the idea that Glu-108 acts in the proton transfer from C2 to C1 in the active site. Although the negatively charged end of R5P requires Rpi to have the most conserved residues, the O3 and O4 atoms of R5P are recognized by Asp-86 and Asp-89 (S-motif) and Lys99 (C-motif) (Fig. 4, B and E). The O3 oxygen atom forms hydrogen bonds with the side chains of Lys-99 (3 Å) and Asp-86 (3.1 Å) and the backbone carbonyl of Thr-30 in the P-motif (2.7 Å) (Fig. 4, B and E). O4 also interacts with Asp-89 (2.7 Å), the backbone amide nitrogen of Gly-102 (3.4 Å) of the C-motif, and the water molecule W1 (2.6 Å). Binding Mode of the C2 Epimer Inhibitor A5P—As for the ttRpi·R5P structure, it was possible to fit an extended ring-opened form of A5P to the electron density with the O1 atom situated in the oxyanion hole (Fig. 4, C and F). The O2 of the inhibitor is recognized similarly to the O2 of R5P forming hydrogen bonds with both the carboxyl oxygens of Glu-108 and the amino nitrogen of the Lys-99 side chain. The binding mode of A5P displays an inverse conformation with respect to R5P so that the interactions with O3 and O4 differ. The O4 and O3 of A5P form hydrogen bond interactions with the side chains of Asp-86 and Asp-89, respectively. Recognition of the Phosphate Group of C2 Epimers, R5P and A5P—The phosphate groups of both C2 epimers, R5P and A5P, bind at identical positions in ttRpi and are recognized by residues of the P-motif. The recognition manner of the phosphate group of the ligands is similar and involves hydrogen bonds to ordered water molecules bound at the same positions in both complex structures (Fig. 4, B and C). The phosphate group of R5P or A5P is situated at the N terminus of the H2 helix (Fig. 2, B and C), and a positive dipole moment from this helix may stabilize the negative charge of the phosphate group. The phosphate group of R5P or A5P forms direct hydrogen bonds with the side" @default.
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• W2146705615 date "2003-12-01" @default.
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• W2146705615 title "Oxyanion Hole-stabilized Stereospecific Isomerization in Ribose-5-phosphate Isomerase (Rpi)" @default.
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• W2146705615 doi "https://doi.org/10.1074/jbc.m309272200" @default.
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